SHORT COMMUNICATION

Collagen coated electrospun polycaprolactone (PCL)with titanium dioxide (TiO2) from an environmentally benignsolvent: preliminary physico-chemical studies for skin substitute

Kajal Ghosal & Sabu Thomas & Nandakumar Kalarikkal &Arumugam Gnanamani

Received: 4 November 2013 /Accepted: 6 March 2014# Springer Science+Business Media Dordrecht 2014

Abstract Fabrication of nanofibers with some biomaterialsbased on natural materials (collagen) through electrospinningis an important area for research. The effect of collagen coatingon polycaprolactone (PCL) nanofiber surfaces was studiedhere. In this work, PCL nanofibers with titanium dioxide(TiO2) nanopowder were used for the development of activewound dressings. We used glacial acetic acid as an environ-mentally benign solvent. The prepared nanofibers were coatedwith collagen by soaking the scaffold in 10 mg/mL and20 mg/ml collagen solution overnight. The samples producedwere subjected to contact angle measurements, SEM, FTIR,andXRD, andmechanical strengthwas determined. Nanofibersin the range of 200–800 nm were produced. The other studyconfirmed the physical interaction between collagen and PCL.The hydrophilicity of PCL nanofibers was increased; this wasconfirmed by observing contact angle values. A hydrophilicsurface on the scaffold is necessary for biomedical applications.FTIR have proved the presence of an amide group on the PCLstructure that facilitates cell adhesion and proliferation. SEMimages have clearly proved the formation of nanofibers as wellas the attachment of collagen to PCL nanofibers. XRD hasshown the crystalline nature of the PCL polymer. PCL canimpart more mechanical strength, although incorporation ofcollagen has decreased the tensile strength to some extent.

Keywords Electrospinning . PCL . Collagen . TiO2.Wound

dressing

Introduction

Open injuries can lead to serious bacterial wound infections,including gas gangrene and tetanus, and these, in turn, maydelay a return to normal activities, and, worse, may lead to alife-threatening illness. Wound infection can become a seriousconcern if injured patients get late definitive care or if thenumber of injured persons exceeds available trauma care ca-pacity. Thus wound management is very essential in order tominimize the aforementioned consequences and treatmentcosts. Since ancient times, a myriad of dressings [1] have beenapplied to cover upwounds. To date, there is a continuous effortto develop a more suitable wound dressing material. A series ofbiodegradable polymer scaffolds are used for wound dressing,tissue engineering, and drug delivery. Polyesters, the mainfamily of synthetic biodegradable polymers, have vast applica-bility in the biomedical field. Most of the biomedical applica-tions include polyglycolide, polylactide, polycaprolactone [2],and their copolymers. Besides these aliphatic polyesters, differ-ent types of synthetic polymers are also being explored [3] bybiomedical researchers with the goal of finding a strong yetflexible polymer scaffold with long durability.

Our approach in this context is to develop a more advancedwound dressing material of a core sheath nanofiber type. Spe-cifically, we intend to obtain the material through anelectrospinning technique [4] with PCL and antimicrobialTiO2 as the core, whereas collagen acts as the sheath. PCL [5]will act as a structural component, which attributes good me-chanical properties, and collagen provides a moist environmentto the wound bed and effectively manages the wound exudates.Collagen, a natural extracellular matrix (ECM), is a componentfound in many tissues such as bone, skin, tendons, ligaments,

K. Ghosal : S. Thomas :N. KalarikkalCentre for Nanoscience and Nanotechnology, Mahatma GandhiUniversity, Kottayam, Kerala, India

A. GnanamaniMicrobiology Division, Central Leather Research Institute (CSIR),Adyar, Chennai, India

K. Ghosal (*)Centre for Nanoscience and Nanotechnology, Mahatma GandhiUniversity, Priyadarsini Hills, Kottayam, Kerala, India 686560e-mail: kaju_3_ju@rediffmail.com

J Polym Res (2014) 21:410DOI 10.1007/s10965-014-0410-y

and other connective tissues. Due to its excellent assembledstructure, abundant availability in nature, and degradability inbiological environments, it has gainedwide application in tissueengineering [6]. The major limitation of collagen is that it haspoor mechanical properties, which may be overcome by blend-ing it with PCL. Incorporation of metal oxide nanoparticles likeTiO2 is used to improve the antimicrobial properties of thedesigned material. Inorganic materials have shown excellentbacterial resistance and thermal stability over organic antibac-terial materials. Due to their nano-scale size, inorganic nano-particles exhibit improved physical, chemical, and biologicalproperties. TiO2 nanostructures have been extensively studiedas antimicrobial agents. Currently there is a growing interest foruse of TiO2 nanoparticles in polymeric scaffolds. Barzegaryet al. [7] have shown antibacterial effects of TiO2.

Materials

Reconstituted Type I Collagen of bovine skin was obtained asa gift from Dr. A. Gnanamani, Central Leather ResearchInstitute, Adyar, Chennai. PCL (Mw 80,000) was obtainedfrom SigmaAldrich, USA. Analytical grade glacial acetic acidwas obtained from SRL,Mumbai. TiO2 nanopowder of 20 nmsize range was purchased from Sigma-Aldrich.

Methods

Preparation of PCL-TiO2 nanofibers with and withoutcoating

All formulation details (F1-F5) are listed in Table 1. Scaffoldswere fabricated using the electrospinning technique. Nanofi-bers of PCL with and without TiO2 nanopowders were pre-pared using glacial acetic acid as a solvent. TiO2 (0.5 % w/vand 1 % w/v) was dissolved in glacial acetic acid at roomtemperature and was stirred for 1 h. The suspension wassonicated for 15 min. For F1, PCL at a concentration of 8 %(w/v) was dissolved in glacial acetic acid. For F2, PCL at aconcentration of 10 % (w/v) was added to 0.5 % (w/v) TiO2

suspension and for F5, PCL at a concentration of 10 % (w/v)was dissolved in 1.0 % (w/v) TiO2 suspension. All solutions

were again stirred for 24 h. These solutions were sonicated for15min before electrospinning. The PCL/TiO2 and control PCLsolutions were taken in a 10 mL syringe fitted with a (23G) flattip metal needle. The flow rate was set under 2.0 mL per hrthrough a syringe pump. A high voltage of around 20.0 kVfrom a high voltage supply (Model AYRA N801, Goldstar,New Delhi, India) was applied between the metal needle tipand a grounded collector at a temperature of 25 °C and 65±5%relative humidity. The gap between needle and collector waskept at 15 cm. Samples F1, F2, and F5 were prepared using theabove procedure. To obtain samples F3 and F4, Collagencoating over the PCL-TiO2 nanofiber scaffold was done byimmersing F2 overnight in a different concentration of colla-gen solutions. The amounts of collagen were 10 mg/mL and20 mg/ml in glacial acetic acid to prepare F3 and F4, respec-tively. Afterward, the constructs were washed three times withpolybutylene succinate (PBS) and kept air-dried [8].

XRD

TheX-raymeasurements of nanofibers (F2 and F5) were carriedout using a Bruker D8 ADVANCE X-ray diffractometer [9]with a scanning rate of 2° per min using Cu–Kα radiation. TheXRD data were collected in the 2θ range of 5°–60°.

Fourier transform infrared (FTIR) spectra

An FTIR spectroscopic analysis of electrospun material wasmade using Spectrum One (Perkin-Elmer, USAmodel). FTIRspectra were recorded in the wave number range of 4,000–400 cm−1 at room conditions.

Contact angle measurement

Biomaterials will come into contact with water, blood, andother body fluids during their use. So it is desirable to checkthem for wettability while intending to produce materials forbiomedical applications such as scaffolds for wound healingor skin regeneration, cellular proliferation, or tissue engineer-ing. This can be done by checking the contact angle which ismade by a liquid on the surface of the electrospun matrix.Here, surface contact angle measurements for all formulationsin the presence of acetic acid were made according to themethods summarized [10] using a contact angle meter(Holmarc Optomechatronics).

Tensile test

For measurement of mechanical strength of the electrospunnanofibers (F1-F5), tensile testing was carried out using auniversal tensile machine (INSTRON 1408) in accordance[11] with American Standards for Testing Methods (ASTM)D882-97; we used a 500 N load cell at a speed of 1 mm/min

Table 1 Formulationdetails on PCL, collagen,and TiO2

S No Collagen 1(mg/ml)

PCL(mg/ml)

TiO2

(%)

F1 0 80 0

F2 0 100 0.5

F3 10 100 0.5

F4 20 100 0.5

F5 0 100 1.0

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on the specimen. The nanofiber matrices were prepared withwidth of 5 mm and gauge length of 20 mm. The thickness ofthe samples was around 0.2 mm.

SEM

Surface morphology investigation of the electrospun fiber wasdone under a scanning electron microscope (SEM)(S3400NSEM, HITACHI). Prior to scanning under theSEM, the samples were sputter coated with gold using a finecoater (E1010, HITACHI) to avoid charging [12]. On the basisof the SEM photographs, the diameters of fibers were ana-lyzed using analysis software Digimizer VR.

Results and discussions

Contact angle measurement

Contact angle measurements have given a clear view aboutthe hydrophilic and hydrophobic surfaces of scaffolds. When

the study was done using a lower concentration of PCL, as forF1, the contact angle was 86°, and for the F2 formulation, thecontact angle increased to 90°. The F2 formulation was coatedwith different concentrations of collagen to get formulationsF3 and F4. When the contact angle study was done, it wasobserved that there was a reduction in the contact angle of upto 45° for F3 and 42° for F4. For F5, the contact angle was 90°again. Collagen coating increases the hydrophilicity of thescaffold. As a result, it traps water molecules, and the contactangle is reduced. So a collagen coating may act as a suitablebiomaterial scaffold for biomedical applications [13]. All con-tact angle values are given in Table 2.

FTIR analysis

Typical bands such as N–H stretching at 3,273 cm−1 for amideA, C–H stretching at 2,919 cm−1 for amide B, C=O stretchingat 1,600–1,700 cm−1 for amide I, N–H deformation at 1,500–1,550 cm−1 for amide II, and N–H deformation at 1,200–1,300 cm−1 for amide III in collagen and PCL/collagen nano-fiber scaffolds were found (Fig. 1). The amide I, II, and IIIband regions of the spectrum are directly related to polypep-tide conformation. The amide I band is a sensitive marker ofpolypeptide secondary structure. There are C–N stretchingvibrations and N–H bending vibrations as minor vibrationmodes of the amide I band. The vibrational frequency of eachC–Obond depends on the strength of the carbonyl oxygen andinteractions between amide units, both of which are influ-enced by local peptide conformation leading to modificationof the secondary structure [8]. Amide groups were observed incollagen and PCL/collagen nanofiber scaffolds. These amide

Table 2 Contact angles and tensile test values for all formulations

S. No Contact angles (°)n=3

Tensile strength(MPa) n=3

F1 86±3.02 1.76±0.30

F2 90±2.84 1.93±0.43

F3 45±2.00 1.81±0.29

F4 42±2.04 1.75±0.45

F5 90±3.33 2.03±0.22

Fig. 1 FTIR images of differentformulations; a collagen b PCL cF3 d F4

J Polym Res (2014) 21:410 Page 3 of 5, 410

groups and carboxyl groups supported fibroblast attachmentand proliferation in skin tissue engineering.

SEM study

Figure 2 displays the SEMmicrographs of electrospunmatricesobtained from PCL and collagen/PCL blends. Fibers are clearlyformed, with globule structures. The diameters of the fiberswere calculated using image analysis software, Digimizer VR .The average diameter was found to be in between 200 and400 nm for the nanofibers of formulation F1. Other formulationdiameters were between 300 and 800 nm. SEM image ofFig. 2(a) clearly showed the formation of fibers, but with beads.It was believed that the concentration of the PCL polymer wasstill too small. Although the concentrations were high enoughto obtain a suitable viscosity, it was too small to initiate a highdegree of entanglement during solvent evaporation in the jet[14]. This entanglement was needed for fiber formation, andlocally, the charge density increased seriously because of sol-vent evaporation. Along with the concentration of polymer,solvent may have some effect on nanofiber formation. Di-electric constant of the solvent is the main parameter whichwill affect nanofiber formation. Sometimes, the presence ofmetallic particles can change the solution conductivity, andfiber formation may be effected. Here as a metallic nanoparti-cle, we have used TiO2 nanopowder, which may change thesolution conductivity and affect fiber formation. SEM imagesof Fig. 2(b) showed the formation of nanofibers without beads.

Collagen coating has shown some cross-linking with the PCLnanofibers, and it was cleary observed in Fig. 2(c) and (d).

XRD

The diffractograms in Fig. 3 show that PCL/TiO2 combina-tions (F2 and F5) exhibit sharp peaks at 22°, which can bedetected on the diffractogram of PCL, typical of a crystallinematerial.

No characteristic peaks of TiO2 were detected, indicating agood mix of TiO2 with the polymer matrix.

Fig. 2 SEM images of differentformulations; a F1 b F2 c F3 d F4

Fig. 3 XRD image of F2 and F5 formulations

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Tensile strength

Incorporation of PCL has increased tensile strength, whereaspresence of collagen has decreased the mechanical strength ofthis scaffold to some extent. Values are given in Table 2.

Conclusion

Electrospinning of PCL with TiO2 and without TiO2

was carried out using glacial acetic acid and producednanofibers with diameter in the range of 200–800 nm.Biomaterials obtained from natural sources such as col-lagen demonstrated the feasibility and efficacy of coat-ing nanofibers of synthetic biomaterials. Improved hy-drophilicity was seen in the collagen-coated nanofibersversus the uncoated ones. The presence of collagen overthe nanofibers showed the presence of an amide groupover PCL fibers, which will help in cell adhesion andproliferation. TiO2 was well dispersed in the PCL scaf-fold, so no characteristics peaks were observed in XRD.The tensile strength of the scaffold decreased to someextent in the presence of collagen. Still, the effects ofprocessing parameters such as needle diameter, polymer con-centration, solvent, metallic particles, and applied voltageneed to be investigated fully in the electrospinning of PCLpolymer.

Acknowledgements This research has been sponsored by the Univer-sity Grants Commission, India under the UGC D. S. Kothari postdoctoralfellowship scheme.

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